The present invention relates generally to the field of arrays and microarrays and, more particularly, to compositions and methods for modifying the solid supports of the arrays and microarrays.
Microarrays having a plurality of polymeric molecules spatially distributed over and stably associated with the surface of a solid support are becoming an increasingly important tool in bioanalysis and related fields. Microarrays of both polypeptides and polynucleotides have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis.
The current methods of manufacturing arrays and microarrays immobilize the polynucleotides at specific sites on solid supports by either synthesizing the polynucleotides at the desired position, or by presynthesizing the polynucleotides and then attaching them to the solid support. U.S. Pat. No. 5,445,934 discloses a method of on-chip synthesis. In this process, a glass slide is derivatized with a chemical species containing a photocleavable protecting group. Selected sites are deprotected by irradiation through a mask. The deprotected sites are then reacted with a DNA monomer containing a photoprotective group. The process of masking, deprotecting, and reacting is repeated for each monomer attached until an array of site-specific polynucleotide sequences is achieved.
Methods for immobilizing pre-synthesized polynucleotides onto solid supports include simple adsorption, ultra violet linking, and covalent attachment. In general, the attachment of unmodified polynucleotides to unmodified solid supports is inefficient. Therefore, the polynucleotides or the solid support has to be modified to enable attachment. Thus, polynucleotides modified with bovine serum albumin adsorb passively to microtiter plates (Southern, E. M. PCT 89/00460), and biotinylated polynucleotides bind tightly to plates or beads that are coated with avidin or streptavidin. In another method, Carrico, et al., U.S. Pat. No. 4,806,546, have described treatment of a nylon support with an alkylating agent to introduce amidine groups onto the surface of the nylon. The derived nylon surface possesses the capacity to noncovalently bind single stranded nucleic acids. The noncovalently bound nucleic acids are then used as probes to detect specific target nucleic acids in solution.
In a different approach, the solid support is modified with a suitable functional group and/or linker. Thus, the solid support is modified to carry an active group, such as hydroxyl, carboxyl, amine, aldehyde, hydrazine, epoxide, bromoacetyl, maleimide, and thiol groups (Lund et al., U.S. Pat. No. 5,474,895) on its surface to which oligonucleotide can be covalently or non-covalently linked. For example, U.S. Pat. No. 5,514,785 to Ness et al. discloses a process for covalently attaching an oligonucleotide to a nylon support. The nylon support is first treated with an amine-containing polymer thereby forming a reactive imidate ester on the surface of the nylon support. The imidate esters on the surface are then reacted with a primary or secondary amine-containing polymer to form amidine residues that are then conjugated with activated polynucleotides. In another method, disclosed in U.S. Pat. No. 6,013,789 to Rampal, polypropylene film is first aminated by a plasma discharge in the presence of ammonia gas, and then contacted with an oligonucleotide having a terminal phosphorimidazolide, whereupon the oligonucleotide becomes covalently linked to the polypropylene film via a phosphoramidate bond.
The immobilized polynucleotides can be used to construct arrays or microarrays for hybridization assays. A typical method of using microarrays involves contacting nucleotide sequences contained in a fluid with the sequences immobilized on the microarrays under hybridization conditions, and then detecting the hybridization complex. The resultant pattern of hybridized nucleic acids provides information regarding the profile of the nucleotide constituents in the sample tested. A widely used method for detecting the hybridization complex in microarrays is by fluorescence. In one method, probes derived from a biological sample are amplified in the presence of nucleotides that have been coupled to a fluorescent label (reporter) molecule so as to create labeled probes, and the labeled probes are then incubated with the microarray so that the probe sequences hybridize to the complementary sequences immobilized on the microarray. A scanner is then used to determine the levels and patterns of fluorescence.
The art methods of immobilizing polynucleotides on solid support usually result in low coupling yields. In addition, the polynucleotides are bound on the flat two-dimensional surface of the substrate, whereas it is thought that binding the polynucleotides within a three-dimensional polymer matrix would enable more efficient hybridization. Thus, there exists a need for methods and procedures for immobilizing polynucleotides to solid support for fabricating arrays and microarrays.
Methods are provided for modifying a solid support by silylating the support with an agent having the formula H2Nxe2x80x94(CH2)nxe2x80x94SiX3 where n is between 1 and 10, and X is independently chosen from OMe, OEt, OPr, Cl, Br, or I, then activating with a crosslinking reagent, followed by reacting with an amine-containing polymer. The support can be further treated with a cross- linking reagent. A plurality of targets may be stably associated with the support and arranged in a defined manner.
Also provided are methods of attaching targets to a solid support. The support is silylating with an agent having the formula H2Nxe2x80x94(CH2)nxe2x80x94SiX3 where n is between 1 and 10, and X is independently chosen from OMe, OEt, Cl, Br, or I, then activating with a crosslinking reagent, followed by reacting with an amine-containing polymer. The support can be further treated with a crosslinking reagent. The support is then contacted with the targets that can optionally have a spacer arm at the 5xe2x80x2-end or the 3xe2x80x2-end of the target.
Also provided is an array of polynucleotides made by modifying a glass slide by silylating with an agent having the formula H2Nxe2x80x94(CH2)nxe2x80x94SiX3 where n is between 1 and 10, and X is independently chosen from OMe, OEt, Cl, Br, or I, then activating with a crosslinking reagent, followed by reacting with an amine-containing polymer. The support can optionally be reacted with crosslinking agent. A plurality of polynucleotides are stably associated with the support in a defined manner.
These and other objectives, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.